Liposomal ciprofloxacin formulations with encapsulated ciprofloxacin nanocrystals

ABSTRACT

Methods of treatment of NTM lung infections using formulations of liposomal ciprofloxacin. Specific liposome formulations and delivery of such for treatment of respiratory tract infections and other medical conditions, and devices and formulations used in connection with such are described.

GOVERNMENT RIGHTS

This invention was made with government support under 1 R43 AI106188-01awarded by National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions ofliposomal ciprofloxacin for inhalation to prevent or treat respiratorytract infections caused by a variety of microorganisms or intracellularpathogens, particularly non tuberculous mycobacteria (NTM).

BACKGROUND OF THE INVENTION

Respiratory tract infections are caused by a variety of microorganisms.Infections which are persistent have a myriad of consequences for thehealth care community including increased treatment burden and cost, andfor the patient in terms of more invasive treatment paradigms andpotential for serious illness or even death. It would be beneficial ifan improved treatment paradigm were available to provide prophylactictreatment to prevent susceptible patients from acquiring respiratorytract infections as well as increasing the rate or effectiveness oferadication for patients already infected with the microorganisms.

Pulmonary infections with non-tuberculosis mycobacteria (NTM) arenotoriously difficult to treat. They exist in the lungs in variousforms, including within macrophages and in biofilms. These locations areparticularly difficult to access with antibiotics. Furthermore, the NTMmay be either in a dormant (termed sessile), or a replicating phase, andan effective antibiotic treatment would target both phases. We havefound, surprisingly, that certain compositions of ciprofloxacin thatinclude ciprofloxacin encapsulated in liposomes are effective in theirantibacterial activity both against NTM harbored in macrophages as wellas NTM that exist dormant in biofilms.

Lung infection from Mycobacterium avium subspecies hominissuis(hereafter referred as M. avium) and Mycobacterium abscessus (hereafterreferred to as M. abscessus) is a significant health care issue andthere are major limitations with current therapies. The incidence ofpulmonary infections by NTM is increasing (Adjemian et al., 2012;Prevots et al, 2010), specifically with M. avium and M. abscessus(Inderlied et al, 1993). About 80% of NTM in US is associated with M.avium (Adjemian et al., 2012; Prevots et al, 2010). M. abscessus, whichis amongst the most virulent types, ranks second in incidence (Prevotset al, 2010). Diseases caused by both mycobacteria are common inpatients with chronic lung conditions, e.g., emphysema, cystic fibrosis,and bronchiectasis (Yeager and Raleigh, 1973). They may also give riseto severe respiratory diseases, e.g., bronchiectasis (Fowler et al,2006). The infections are from environmental sources and causeprogressive compromising of the lung. Current therapy often fails onefficacy or is associated with significant side-effects. M. aviuminfection is usually treated with systemic therapy with a macrolide(clarithromycin) or an azalide (azithromycin) in combination withethambutol and amikacin. Oral or IV quinolones, such as ciprofloxacinand moxifloxacin, can be used in association with other compounds(Yeager and Raleigh, 1973), but higher intracellular drug levels need tobe achieved for maximal efficacy. Oral ciprofloxacin has clinicalefficacy against M. avium only when administered in combination with amacrolide or an aminoglycoside (Shafran et al 1996; de Lalla et al,1992; Chiu et al, 1990). Studies in vitro and in mouse suggest that thelimited activity of oral ciprofloxacin alone is related to the inabilityof ciprofloxacin to achieve bactericidal concentrations at the site ofinfection (Inderlied et al, 1989); the minimum inhibitory concentration(MIC) of 5 μg/ml versus the clinical serum Cmax of 4 μg/ml explains thelimited efficacy in experimental models and in humans (Inderlied et al,1989). M. abscessus is often resistant to clarithromycin. IVaminoglycosides or imipenem need to be applied, which often are the onlyavailable therapeutic alternatives, and these carry the potential forserious side-effects, as well as the trauma and cost associated with IVadministration. Clofazimine, linezolid, and cefoxitin are also sometimesprescribed, but toxicity and/or the need for IV administration limit theuse of these compounds. Thus, the available therapies have significantdeficiencies and improved approaches are needed.

Recent studies also showed that both M. avium and M. abscessusinfections are associated with significant biofilm formation (Bermudezet al, 2008; Carter et al, 2003): deletion of biofilm-associated genesin M. avium had impact on the ability of the bacterium to form biofilmand to cause pulmonary infection in an experimental animal model(Yamazaki et al, 2006).

Ciprofloxacin is a broad-spectrum fluoroquinolone antibiotic that isactive against several other types of gram-negative and gram-positivebacteria and is indicated for oral and IV treatment of lower respiratorytract infections. It acts by inhibition of topoisomerase II (DNA gyrase)and topoisomerase IV, which are enzymes required for bacterialreplication, transcription, repair, and recombination. This mechanism ofaction is different from that for penicillins, cephalosporins,aminoglycosides, macrolides, and tetracyclines, and therefore bacteriaresistant to these classes of drugs may be susceptible to ciprofloxacin.There is no known cross-resistance between quinolones—the class ofantimicrobials that ciprofloxacin belongs to—and other classes ofantimicrobials.

Despite its attractive antimicrobial properties, ciprofloxacin doesproduce bothersome side effects, such as GI intolerance (vomiting,diarrhea, abdominal discomfort), as well as dizziness, insomnia,irritability and increased levels of anxiety. There is a clear need forimproved treatment regimes that can be used chronically, withoutresulting in these debilitating side effects.

Delivering ciprofloxacin as an inhaled aerosol has the potential toaddress some of these concerns by compartmentalizing the delivery andaction of the drug in the respiratory tract, which is the primary siteof infection. Currently there is no aerosolized form of ciprofloxacinwith regulatory approval for human use, capable of targeting antibioticdelivery direct to the area of primary infection. In part this isbecause the poor solubility and bitterness of the drug have inhibiteddevelopment of a formulation suitable for inhalation; many patients withairway disease may cough or bronchoconstrict when inhaling antibioticswhich are not encapsulated in liposomes (Barker et al, 2000).Furthermore, the tissue distribution of ciprofloxacin is so rapid thatthe drug residence time in the lung is too short to provide additionaltherapeutic benefit over drug administered by oral or IV routes(Bergogne-Bérézin E, 1993).

The therapeutic properties of many drugs are improved by incorporationinto liposomes. Phospholipid vehicles as drug delivery systems wererediscovered as “liposomes” in 1965 (Bangham et al., 1965). The generalterm “liposome” covers a variety of structures, but all consist of oneor more lipid bilayers enclosing an aqueous space in which hydrophilicdrugs, such as ciprofloxacin, can be encapsulated. Liposomeencapsulation improves biopharmaceutical characteristics through anumber of mechanisms including altered drug pharmacokinetics andbiodistribution, sustained drug release from the carrier, enhanceddelivery to disease sites, and protection of the active drug speciesfrom degradation. Liposome formulations of the anticancer agentsdoxorubicin (Myocet®/Evacet®, Doxyl®/Caelyx®), daunorubicin (DaunoXome®)the anti-fungal agent amphotericin B (Abelcet®, AmBisome®, Amphotec®)and a benzoporphyrin (Visudyne®) are examples of successful productsintroduced into the US, European and Japanese markets over the last twodecades. Recently a liposomal formulation of vincristine (Marqibo®) wasapproved for an oncology indication. The proven safety and efficacy oflipid-based carriers make them attractive candidates for the formulationof pharmaceuticals.

Therefore, in comparison to the current ciprofloxacin formulations, aliposomal ciprofloxacin aerosol formulation should offer severalbenefits: 1) higher drug concentrations, 2) increased drug residencetime via sustained release at the site of infection, 3) decreased sideeffects, 4) increased palatability, 5) better penetration into thebacterial biofilms, and 6) better penetration into the cells infected bybacteria.

In one example of the current invention, the liposomes encapsulatingciprofloxacin are unilamellar vesicles (average particle size 75-120nm). Ciprofloxacin is released slowly from these liposomes with ahalf-life of about 10 hours in the lung (Bruinenberg et al, 2010 b),which allows for once-a-day dosing. Further, studies with a variety ofliposome compositions in in vitro and murine infection models showedthat liposomal ciprofloxacin is effective against several intracellularpathogens, including M. avium. Inhaled liposomal ciprofloxacin is alsoeffective in treating Pseudomonas aeruginosa (PA) lung infections inpatients (Bilton et al, 2009 a, b, 2010, 2011; Bruinenberg et al, 2008,2009, 2010 a, b, c, d, 2011; Serisier et al, 2013).

Compared to approved doses of oral and IV ciprofloxacin, liposomalciprofloxacin formulations delivered by inhalation into the airwaysachieve much greater concentrations in the respiratory tract mucosa andwithin macrophages with resulting improvement of clinical efficacy: 2hours post-inhalation of a therapeutic dose of our liposomalciprofloxacin in patients, the concentration of ciprofloxacin in thesputum exceeded 200 μg/ml, and even 20 hours later (2 hours prior to thenext dose), the concentration was >20 μg/ml, well above the minimuminhibitory concentration above for resistant mycobacteria (breakpoint of˜4 μg/ml (Bruinenberg 2010b). Since the liposomes containingciprofloxacin are avidly ingested by macrophages, the ciprofloxacin isbrought into close proximity to the intracellular pathogens, thusfurther increasing anti-mycobacterial concentration and thus should leadto improved efficacy of the inhaled liposomal formulation compared toother forms of ciprofloxacin. We therefore believe that even highlyresistant NTM may be suppressed with our inhaled liposomalciprofloxacin. This is significant because M. avium and M. abscessusresistance to antibiotics is common due to long-term use of systemicantibiotics in these patients. Our clinical experience with P.aeruginosa (PA) also shows that there is no apparent emergence ofresistance following inhaled liposomal ciprofloxacin therapy: in fact,even those patients who also had resistant strains initially, respondedwell to therapy (Serisier et al., 2013). This is likely due to thepresence of sustained overwhelming concentrations of ciprofloxacin.Furthermore, the experience with other anti-pseudomonal drugs tobramycinand colistimethate in patients with cystic fibrosis is that evenpatients with resistant strains of PA respond clinically well to theinhaled form of the drugs (Fiel, 2008).

Several in vitro studies have demonstrated that liposomal ciprofloxacinis efficacious against intracellular pathogens: 1) In human peripheralblood monocytes/macrophages, liposomal ciprofloxacin tested overconcentrations from 0.1 to 5 μg/ml caused concentration-relatedreductions in intracellular M. avium-M. intracellulare complex (MAC)colony forming units (CFU) compared to free drug at the sameconcentrations (Majumdar et al, 1992); 2) In a murine macrophage-likecell line J774, liposomal ciprofloxacin decreased the levels of cellassociated M. avium up to 43-fold and these reductions were greater thanfor free ciprofloxacin (Oh et al, 1995).

Once M. avium or M. abscessus infect monocytes/macrophages, theinfection can then spread to the lungs, liver, spleen, lymph nodes, bonemarrow, and blood. There are no published studies on the efficacy ofliposomal ciprofloxacin against M. avium or M. abscessus in animalmodels.

A few in vivo studies have demonstrated that liposomal ciprofloxacin isefficacious against the intracellular pathogen, F. tularensis: Efficacyof liposomal ciprofloxacin delivered to the lungs by inhalation orintranasal instillation against inhalational tularemia (F. tularensislive vaccine strain (LVS) and Schu S4) in mice, was demonstrated with aslittle as a single dose of liposomal ciprofloxacin providing 100%protection post-exposure, and even effective post-exposure treatment foranimals that already had significant systemic infection (Blanchard etal, 2006; Di Ninno et al, 1993; Conley et al, 1997; Hamblin et al, 2011;Hamblin et al, 2014; Wong et al, 2003). These studies also found thatinhaled liposomal ciprofloxacin was superior to both inhaled and oralunencapsulated ciprofloxacin.

In contrast, a) free ciprofloxacin was inferior to liposomalciprofloxacin in macrophage models of mycobacterial infections (Majumdaret al, 1992; Oh et al, 1995); b) free ciprofloxacin alone delivered tothe lungs had inferior efficacy to free ciprofloxacin when tested inmurine models of F. tularensis infection (Conley et al, 1997; Wong etal, 2003), as it is rapidly absorbed into the blood stream. Aformulation made up of both free and liposomal ciprofloxacin combinesthe potential advantages of an initial transient high concentration offree ciprofloxacin to increase Cmax in the lungs, followed by the slowrelease of ciprofloxacin from the liposomal component, as demonstratedin non-CF bronchiectasis patients by Aradigm (Cipolla et al, 2011;Serisier et al, 2013). The free ciprofloxacin component also has adesirable immunomodulatory effect (U.S. Pat. Nos. 8,071,127, 8,119,156,8,268,347 and 8,414,915).

Further, liposomal ciprofloxacin injected parenterally activatesmacrophages, resulting in increased phagocytosis, nitric oxideproduction, and intracellular microbial killing even at sub-inhibitoryconcentrations, perhaps via immunostimulatory effects (Wong et al,2000). The ciprofloxacin-loaded macrophages may migrate from the lungsinto the lymphatics to treat infections in the liver, spleen, and bonemarrow—as suggested by the systemic effects of pulmonary-delivered CFIin tularemia (Di Ninno et al, 1993; Conley et al, 1997; Hamblin et al,2011; Hamblin et al, 2014; Wong et al, 2003). Liposome-encapsulatedantibiotics are also known to better penetrate bacterial films formed byP. aeruginosa in the lungs (Meers et al, 2008). However, it has not beendemonstrated before that antibiotic-loaded liposomes in general, orliposomally encapsulated ciprofloxacin, would be able to penetratebiofilms formed by mycobacteria, and specifically by NTM in the lung.The anti-mycobacterial and immunomodulatory effects of the newformulations delivered to the lungs, may therefore provide a betteralternative to the existing treatments for patients infected with M.avium or M. abscessus, or provide an adjunct for incrementalimprovements if the antibiotic preparation is effective against theseorganisms that are planktonic, as well as in the biofilms and withinmacrophages. It is further required that the antibiotic treatment iswell tolerated and safe when given by inhalation. Since the currentantibiotic treatment options often cause serious systemic side-effects,it is desirable for the new treatment to have less toxic antibiotics andto minimize their concentration in the circulation to avoid systemicside effects.

A study of liposomal ciprofloxacin demonstrated high uptake by alveolarmacrophages in animals, which is presumably the reason for the highlyeffective post-exposure prophylaxis and treatment of inhalationaltularemia in mice. Although the plasma levels of ciprofloxacin were lowfollowing respiratory tract administration of our liposomalciprofloxacin, a reduction of the tularemia infection from the liver,spleen, tracheobronchial lymph nodes, as well as the lungs, was observedsuggesting that the alveolar macrophages loaded with liposomalciprofloxacin migrate from the lungs via lymph into the liver, spleenand lymph nodes (F. tularensis CFU levels in bone marrow and blood werenot measured) (Conley et al, 1997).

SUMMARY OF THE INVENTION

The current clinical paradigm is to treat patients with M. avium or M.abscessus lung infections with combination therapy given orally or byIV. But the current treatment methods have many issues, as discussedabove, and do not have good efficacy. The formulation of the inventionis an inhaled liposomal ciprofloxacin formulation with improved efficacyfor NTM patients and allows for once daily inhalation that may becombined with other treatments. Inhaled liposomal ciprofloxacin of theinvention provides for systemic therapy against M. avium disseminatedinfection, working in synergy with other antibiotics and achieving highconcentrations in the lung. The transport of the macrophage-ingestedliposomal ciprofloxacin via the lymphatics to the spleen and liver mayoccur, as observed in the murine model of F. tularensis.

The method of treating a non-tuberculous mycobacteria infectioncomprises:

-   -   aerosolizing a dose of a formulation to create aerosolized        particles having an aerodynamic diameter in a range of 1 micron        to 12 microns, or 2 microns to 10 microns, or 4 microns to 8        microns; and    -   inhaling the aerosolized particles into the patient's lungs        about once a day for about seven to 56 days or until the        infection is eradicated.

The formulation is comprised of an anti-infective drug which may beciprofloxacin. The formulation may include a liquid carrier which may beliquid drug or an inert liquid such as water or ethanol. The carrier mayhave drug dissolved or dispersed therein.

The carrier will have liposomes dispersed therein and the liposomesencapsulate nanocrystals of an anti-infective pharmaceutically activedrug which may be ciprofloxacin. The liposomes are comprised of a lipidbilayer which initially encapsulates a solution of carrier and drug,followed by freezing to −20° C. to −80° C., followed by thawing wherebydrug nanocrystals remain inside the liposome bilayer.

The liposomes may be comprised of a cryopreservative and a surfactant.The cryopreservative may be a polyol, e.g. sucrose or trehalose. Thesurfactant is a nonionic detergent which may be polysorbate 20 and/orBRIJ 20.

The invention further includes use of a formulation to treatnon-tuberculous mycobacteria wherein the formulation is produced by aparticular method whereby the drug such as ciprofloxacin is dissolved inan aqueous solution at a concentration in a range of 25 mg/mL or more,50 mg/mL or more, 100 mg/mL or more, 200 mg/mL or more and encapsulatedinto a lipid bilayer of liposomes. The liposomes are then includedwithin a solution which may include an anti-infective which may be thesame or different from the anti-infective compound encapsulated withinthe liposomes and as such may be ciprofloxacin. The formulation isfrozen such as being frozen at very low temperatures in the range of−20° C. to −80° C. The frozen formulation may be maintained frozen overlong periods of time for storage such as one week or more, one month ormore, one year or more or may be immediately rethawed for use. Uponrethawing drug inside of the liposomes forms nanocrystals. Uponadministration the drug dissolved in the solvent carrier surrounding theliposomes provides for immediate release of drug followed by a drugbeing released when the liposomes dissolve in the lung followed by anadditional release of drug when the nanocrystals dissolve. Theformulation provides for controlled release of an anti-infective drugsuch as ciprofloxacin over a long period of time in the lungs therebymaking it possible to effectively eradicate infections which occur as abiofilm.

The combination of the encapsulation of ciprofloxacin in liposomes withdirect delivery of the formulation to the lungs makes these treatmentsfundamentally different from oral and parenteral products ofciprofloxacin and other antibiotics in terms of biodistribution,pharmacokinetics, as well as improved safety and efficacy. Theliposome-encapsulated ciprofloxacin is delivered at very highconcentrations directly to the respiratory tract where it resides over aprolonged period of time, during which ciprofloxacin is slowly releasedfrom the liposomes to the site of infection in the lung, and with lowersystemic exposure compared to oral or IV ciprofloxacin.

The size and composition of the liposomal ciprofloxacin formulations aredesigned to facilitate uptake by the macrophages in the lung. The mostimportant feature is that the formulation should be robust to thenebulization process so that the liposomes retain their size andencapsulation characteristics. If the liposomes are not robust toaerosolization, then there could be loss of encapsulated drug, or achange in the liposome size or surface characteristics. Either of thesechanges, or others that have not been described, might lead to loweruptake of the liposomes by macrophages. The liposomes that lose aportion of their encapsulated drug during nebulization oraerosolization, even if they are taken up by the liposomes with the sameefficiency as uncompromised liposomes, now have less encapsulated drugand thus a lower payload to treat the infectious agent inside themacrophages and in biofilms thereby reducing the efficacy of treatment.

One particular composition of liposomes, which are covered by thisinvention, are relatively uncompromised by the nebulization process andhave been described in U.S. Pat. Nos. 8,071,127, 8,119,156, 8,268,347and 8,414,915. Those patents describe an aerosolizable, bi-phasicaerosol of inhaled droplets or particles. The droplets or particlescomprise a free drug (e.g., an anti-infective compound) in which drug isnot encapsulated and which may be ciprofloxacin. The particles furthercomprise a liposome which encapsulates a drug such as an anti-infectivecompound which also may be ciprofloxacin. The free and liposomeencapsulated drug are included within a pharmaceutically acceptableexcipient which is formulated for aerosolized delivery. The particlesmay further include an additional therapeutic agent which may be freeand/or in a liposome and which can be any pharmaceutically active drugwhich is different from the first drug.

Other liposome compositions include those which are modified bynebulization, leading to changes in vesicle size, or drug encapsulation,or both. These include liposomes containing drugs such as amikacin thathave been described in U.S. Pat. Nos. 8,226,975, 8,642,075, 8.673.348,8,673,349, and U.S. Patent applications: 2007196461, 20130028960,20130052260, 20130064883, 20130071469, 20130087480, 20130330400,20140072620. US Patent application 20130330400 specifically describes aliposomal formulation of amikacin that is compromised by nebulizationsuch that only 58 to 73% of the drug remains encapsulated after exposureto nebulization. In this application, US Patent application 20130330400,the mean vesicle size was also affected by the nebulization process witha reduction from a mean of 285 nm prior to nebulization to 265 nm afternebulization (range: 249 to 289 nm). US Patent application 20140072620also describes a liposomal amikacin formulation that degrades to 60%encapsulated and 40% free drug after nebulization.

Liposomes used in connection with the present invention retain 80% ormore, and preferably 90% or more, and most preferably 95% or more of theencapsulated drug after nebulization relative to that which wasencapsulated prior to nebulization. If significant amounts of the drugare lost from the liposomes during nebulization, for example, greaterthan 20% of the encapsulated drug, then during liposome uptake by themacrophages, assuming that was designed into the feature set of theliposomes, there will be less encapsulated drug to be released into themacrophages, thereby compromising in vivo efficacy. This is a keyelement in the ability of the liposomal formulation to be active againstthe intracellular infections. Even if one conducts in vitro or in vivostudies and is able to demonstrate efficacy in those models, in the realworld situation the formulation will be nebulized first, or otherwiseaerosolized, so there is no assurance about the validity of the resultsin the model unless the formulation was applied as an aerosol (which isnot typical), or other studies have been conducted to verify that theintegrity of the liposomes is maintained following aerosolization.

The alveolar macrophages are targeted by M. avium and M. abscessus(Jordao et al, 2008) and other mycobacteria species as well. Themacrophages avidly ingest both the liposomal ciprofloxacin and themycobacteria, bringing both into close proximity within the phagosomes.This increase in the bioavailability at the infected target, thealveolar macrophage cells in the lung, provides improved efficacy versussystemically delivered ciprofloxacin or other anti-mycobacterial agents.The sustained-release of ciprofloxacin from the liposomes furtherincreases the ratio of the area under the curve to MIC (AUC/MIC) in thelungs and macrophages, in particular, and may enable once-a-day dosing.The administration of these formulations provides a lower incidence ofrelapse and reduced adverse systemic effects.

An aspect of the invention is an aerosol of inhaled droplets orparticles. The droplets or particles comprise a free drug (e.g., ananti-infective compound) in which drug is not encapsulated and which maybe ciprofloxacin. The particles further comprise a liposome whichencapsulates a drug such as an anti-infective compound which also may beciprofloxacin. The free and liposome encapsulated drug are includedwithin a pharmaceutically acceptable excipient which is formulated foraerosolized delivery. The particles may further include an additionaltherapeutic agent which may be free and/or in a liposome and which canbe any pharmaceutically active drug which is different from the firstdrug.

Another aspect of the invention is a formulation comprising liposomeswhich are delivered via an aerosol to the lungs of a human patient withan NTM infection, or to prevent an NTM infection, the liposomescomprising free and encapsulated ciprofloxacin. The liposomes may beunilamellar or multilamellar. The aerosolization can be achieved bynebulization, including jet nebulization or mesh nebulization. Theencapsulated ciprofloxacin is in liposomes which are robust to thenebulization process and maintain their encapsulation state to greaterthan 90% following nebulization, and preferably to greater than 95%following nebulization.

A third aspect of the invention is a method for treating intracellularinfections in a patient, the method comprising administering aformulation comprising the anti-infective; e.g., ciprofloxacin,encapsulated in liposomes to the patient. The formulation is preferablyadministered by inhalation to the patient, and more preferably bynebulization. The intracellular infections may represent NTM infectionsincluding M. abscessus, M avium, M. avium complex, (MAC) (M. avium andM. intracellulare), M. Bolletii, M. chelonae, M. ulcerans, M. xenopi, M.kansasii, M. fortuitum complex (M. fortuitum and M. chelonae) or M.marinum infections.

A fourth aspect to the invention is the ability of the liposomalanti-infective formulation, preferably a liposomal ciprofloxacinformulation, after aerosolization and delivery to the airways and deeplung, to be taken up by macrophages and possess the ability to kill bothreplicating and non-replicating (sessile) mycobacteria. For successfultreatment of NTM, it is essential that the treatment kills bothreplicative and dormant (or sessile) mycobacteria because both forms arefound in mycobacterial infections.

The fifth aspect of the invention is that for the treatment to bemaximally effective, the antibiotic formulation also needs to be able topenetrate the biofilm formed by the mycobacteria.

The sixth aspect of the invention is that the antibiotic in a suitablevehicle is not only able to penetrate the biofilm but also to haveefficacy against both sessile (dormant) and replicating mycobacteria.

Another aspect of the invention is that the antibiotic does not enhancethe formation of mycobacterial biofilms in the lung. In particular, M.avium forms biofilm, a property in mice that is associated with lunginfection via aerosol. Incubation of M. avium with two antibiotics foundin the environment, streptomycin and tetracycline, results in anincrease, not decrease, in the biofilm formation. Other antibiotics,including ampicillin, moxifloxacin, rifampicin and TMP/SMX had no effecton biofilm; i.e., they were not able to kill the M. avium. Moxifloxacinis a fluoroquinolone, like ciprofloxacin. Accordingly, it is surprisingthat specific liposomal ciprofloxacin formulations of the invention areeffective at killing mycobacteria in biofilm. Note that even if anantibiotic is able to kill all of the planktonic phenotype ofmycobacteria, both planktonic and sessile bacteria are able to establishinfection equally, ensuring that the remaining sessile bacteria willreinfect the host. Many patients with chronic lung conditions aretreated for infections caused by many pathogens with antibiotics, suchas aminoglycosides or tetracyclines. Therefore, in the situation that M.avium is colonizing an individual receiving an antibiotic, either forprophylaxis or therapy, it may result in the production of increasedamounts of biofilm and further establishment of the infection.

According to another aspect of the present invention, a formulationcomprising both a free and encapsulated anti-infective provides aninitially high therapeutic level of the anti-infective in the lungs,while maintaining a sustained release of anti-infective over time, toovercome the barrier to eradicate the difficult to treat biofilmbacteria. The intent of the immediate-release anti-infective; e.g.,ciprofloxacin, is thus to rapidly increase the antibiotic concentrationin the lung to therapeutic levels around the difficult to eradicatebiofilm bacteria to address the challenges of lower diffusion rate ofantibiotic to and within the biofilm. The sustained-releaseanti-infective; e.g., ciprofloxacin, serves to maintain a therapeuticlevel of antibiotic in the lung thereby providing continued therapy overa longer time frame, increasing efficacy, reducing the frequency ofadministration, and reducing the potential for resistant colonies toform. The sustained release of the anti-infective from liposomes of theinvention ensures that the anti-infective agent never falls below thesub-inhibitory concentration and so reduces the likelihood of formingresistance to the anti-infective.

Although ciprofloxacin is a particularly useful anti-infective in thisinvention, there is no desire to limit this invention to ciprofloxacin.Other antibiotics or anti-infectives can be used such as those selectedfrom the group consisting of: an aminoglycoside, a tetracycline, asulfonamide, p-aminobenzoic acid, a diaminopyrimidine, a quinolone, abeta-lactam, a beta-lactam and a beta-lactamase inhibitor,chloramphenicol, a macrolide, penicillins, cephalosporins, linomycin,clindamycin, spectinomycin, polymyxin B, colistin, vancomycin,bacitracin, isoniazid, rifampin, ethambutol, ethionamide, aminosalicylicacid, cycloserine, capreomycin, a sulfone, clofazimine, thalidomide, apolyene antifungal, flucytosine, imidazole, triazole, griseofulvin,terconazole, butoconazole ciclopirax, ciclopirox olamine, haloprogin,tolnaftate, naftifine, terbinafine, or any combination thereof.

Antibiotics that are effective against both dormant (sessile) andreplicating bacteria are preferred.

An aspect of the invention is a formulation used for, or a method oftreating a non-tuberculous mycobacteria infection, comprising:

-   -   aerosolizing a dose of a formulation to produce aerosolized        particles having an aerodynamic diameter in a range of from 1        micron to 12 microns;    -   inhaling the aerosolizing particles into lungs of a patient,    -   wherein the aerosolized formulation is comprised of a liquid        carrier comprising free ciprofloxacin at a concentration of 20        mg/mL to 80 mg/mL of ciprofloxacin in solution, liposome        unencapsulated ciprofloxacin in solution and ciprofloxacin as        nanocrystals encapsulated inside the liposomes;    -   wherein the aerosolized particles have an aerodynamic diameter        of two microns to eight microns, the liposomes have a diameter        of 20 nanometers to 1 micron and the nanocrystals have        dimensions of 10 nanometers or less;    -   wherein the ciprofloxacin is present in the solution in a        concentration of 30 mg/mL to 70 mg/mL; or    -   wherein the ciprofloxacin is present in the solution at a        concentration of 40 mg/mL to 60 mg/mL;    -   wherein the liposomes are unilamellar and maintain structural        integrity at a level of 90% or more after aerosolizing; and    -   repeating the aerosolizing and inhaling once each day over a        period of at least seven days; or    -   wherein the repeating is carried out each day, once a day over a        period of seven days to fifty six days, and wherein the        infection is in a biofilm.

Another aspect of the invention is a use or method as described abovefurther comprising:

-   -   assaying the patient for infection;    -   continuing the repeating aerosol administration when the patient        tests positive and discontinuing when the patient tests        negative;    -   wherein the liposomes are comprised of a cryopreservative and a        surfactant;    -   wherein 95% or more of the liposomes maintain structural        integrity and continue to encapsulate nanocrystals of        ciprofloxacin after aerosolizing the formulation; or    -   wherein 98% or more of the liposomes maintain structural        integrity and continue to encapsulate nanocrystals of        ciprofloxacin after aerosolizing the formulation.

Yet another aspect of the invention is a use of a formulation intreatment of non-tuberculous mycobacteria infection, wherein theformulation comprises:

-   -   liposomes wherein the liposomes comprise:    -   a lipid bilayer;    -   a cryopreservative;

and

-   -   nanocrystals of a pharmaceutically active anti-infective drug        surrounded by the lipid bilayer wherein the nanocrystals have        dimensions of 100 nm or less;    -   wherein the cryopreservative is a polyol;    -   wherein the polyol is sucrose or trehalose;    -   wherein the liposomes further comprise a surfactant.

The use of the invention as described above further comprising:

-   -   a pharmaceutically acceptable carrier; or    -   a pharmaceutically active anti-infective drug dissolved in the        carrier; or    -   a liquid anti-infective drug in which the liposomes are        dispersed.

The use as described above further includes a use wherein theanti-infective drug is selected from the group comprising a quinolone, asulfonamide, an aminoglycoside, a tetracycline, para-aminobenzoic acid,a diaminopyrimidine, a beta-lactam, a beta-lactam and a beta-lactamaseinhibitor, chloramphenicol, a macrolide, lincomycin, clindamycin,spectinomycin, polymyxin B, colistin, vancomycin, bacitracin, isoniazid,rifampin, ethambutol, ethionamide, aminosalicylic acid, cycloserine,capreomycin, a sulfone, clofazimine, thalidomide, polyene antifungal,flucytosine, imidazole, triazole, griseofulvin, terconazole,butoconazole ciclopirax, ciclopirox olamine, haloprogin, tolnaftate,naftifine, terbinafine and combinations thereof; and

-   -   wherein the lipid bilayer is comprised of a lipid selected from        the group consisting of fatty acids; lysolipids; sphingolipids;        sphingomyelin; glycolipids; glucolipids; glycosphingolipids;        palmitic acid; stearic acid; arachidonic acid; oleic acid;        lipids bearing sulfonated mono-, di-, oligo- or polysaccharides;        lipids with ether and ester-linked fatty acids, polymerized        lipids, diacetyl phosphate, stearylamine, cardiolipin,        phospholipids, synthetic phospholipids with asymmetric acyl        chains; and lipids bearing a covalently bound polymer; and    -   wherein the liposome comprises a phospholipid selected from the        group consisting of phosphatidylcholines,        lysophosphatidylcholines, phosphatidylethanolamines,        phosphatidylinositols, phosphatidylglycerols, phosphatidic acid,        phosphatidylserines, and mixtures thereof; wherein said        phospholipid is provided in admixtures with a modifying agent        selected from the group consisting of cholesterols, stearyl        amines, stearic acid, tocopherols, and mixtures thereof; and        wherein the liposomes are unilamellar or multilamellar; and    -   wherein the liposomes are comprised of HSPC and cholesterol; and    -   wherein the lipid bilayer is comprised of HSPC and cholesterol;    -   the cryopreservation is selected from the group consisting of        sucrose and trehalose;    -   the surfactant is selected from the group consisting of        polysorbate 20 and BRIJ 20; and    -   the drug is ciprofloxacin.

The invention further include any use or formulation as described herewherein liposomes are comprised of a polyol and aphosphatidylcholine-enriched phospholipids present at a ratio between1:10 to 10:1 (w/w); and/or

-   -   wherein liposomes are comprised of a polyol and a        phosphatidylcholine-enriched phospholipids present at a ratio        between 1:1 to 5:1 (w/w); and/or    -   wherein the surfactant is present in an amount of between 0.01%        to 1%; and/or    -   wherein the surfactant is present in an amount between 0.05% to        0.4%; and/or    -   wherein the infection is an infection on a biofilm in the lung        of the patient and the liposomes release drug over a period of        time and at a rate effective in treating a biofilm infection.

The invention also includes any use for or method of treating anantibiotic resistant infection in a patient, comprising:

-   -   aerosolizing a formulation comprising free ciprofloxacin and        ciprofloxacin encapsulated in liposomes; and    -   inhaling the aerosol into the patient's lungs whereby 90% or        more of the liposomes maintain structural integrity after being        aerosolized,    -   wherein the antibiotic resistant infection comprises        microorganisms in a biofilm or microorganisms engulfed in        macrophage;    -   wherein the infection is an infection of microorganisms in a        biofilm; and/or    -   wherein the infection is an infection of microorganisms engulfed        in macrophage in a planktonic state; and/or    -   wherein the infection is an infection of microorganisms selected        from the group consisting of mycobacteria, P. aeruginosa and F.        tularensis; and/or    -   the liposomes comprise a cryopreservative and a surfactant and        have an average particle size of about 75 nm to about 120 nm and        are unilamellar; and/or    -   the liposomes are comprised of cholesterol and hydrogenated soy        phosphatidyl-choline (HSPC)-a semi-synthetic fully hydrogenated        derivative of nature soy lecithin at a ratio of about 30 to 70        (plus or minus 10%); and/or    -   the formulation further comprising an excipient suitable for        pulmonary delivery comprised of sodium acetate and an isotonic        buffer; and/or    -   90% or more of the liposomes maintain integrity when aerosolized        and after contacting lung tissue provide a ciprofloxacin release        rate of 0.5% to 10% per hour; and/or    -   95% or more of the liposomes maintain integrity when aerosolized        and after contacting lung tissue provide a ciprofloxacin release        rate of 1% to 8% per hour; and/or    -   the liposomes comprise cholesterol and hydrogenated soy        phosphatidyl-choline (HSPC) at a ratio of 29.4 to 70.6, and are        unilamellar and wherein 98% or more of the liposomes maintain        integrity when aerosolized, and provide a ciprofloxacin release        rate of 2% to 6% per hour.

The invention includes a formulation use or method described herewherein:

-   -   the liposomes are further comprised of 0.1 to 0.3% polysorbate        20, and 200 to 400 mg/mL sucrose; and/or    -   the aerosolizing and inhaling are repeated once each day over a        period of seven days or more; and/or    -   the aerosolizing and inhaling are repeated once each day over a        period of seven days to fifty-six days; and/or    -   the formulation comprises 50 mg to 500 mg of ciprofloxacin;        and/or    -   the formulation comprises 75 mg to 300 mg of ciprofloxacin;        and/or    -   the formulation is nebulized and comprises 150 mg of        ciprofloxacin.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the formulations and methodology as more fully describedbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects and embodiments of the invention are best understood from thefollowing detailed description when read in conjunction with theaccompanying drawings. It is emphasized that, according to commonpractice, the various features of the drawings are not to-scale. On thecontrary, the dimensions of the various features are arbitrarilyexpanded or reduced for clarity. Included in the drawings are thefollowing figures:

FIG. 1 is a manufacturing flow chart of liposomal ciprofloxacin forinhalation (HSPC/Chol-10 L Batch).

DETAILED DESCRIPTION OF THE INVENTION

Before the present method of formulating ciprofloxacin-encapsulatedliposomes and delivery of such for prevention and/or treatment of NTMinfections and other medical conditions, and devices and formulationsused in connection with such are described, it is to be understood thatthis invention is not limited to the particular methodology, devices andformulations described, as such methods, devices and formulations may,of course, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aformulation” includes a plurality of such formulations and reference to“the method” includes reference to one or more methods and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

As used herein, anti-infective refers to agents that act againstinfections, such as bacterial, viral, fungal, mycobacterial, orprotozoal infections.

Anti-infectives covered by the invention include but are not limited toquinolones (such as nalidixic acid, cinoxacin, ciprofloxacin andnorfloxacin and the like), sulfonamides (e.g., sulfanilamide,sulfadiazine, sulfamethaoxazole, sulfisoxazole, sulfacetamide, and thelike), aminoglycosides (e.g., streptomycin, gentamicin, tobramycin,amikacin, netilmicin, kanamycin, and the like), tetracyclines (such aschlortetracycline, oxytetracycline, methacycline, doxycycline,minocycline and the like), para-aminobenzoic acid, diaminopyrimidines(such as trimethoprim, often used in conjunction with sulfamethoxazole,pyrazinamide, and the like), penicillins (such as penicillin G,penicillin V, ampicillin, amoxicillin, bacampicillin, carbenicillin,carbenicillin indanyl, ticarcillin, azlocillin, mezlocillin,piperacillin, and the like), penicillinase resistant penicillin (such asmethicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin and thelike), first generation cephalosporins (such as cefadroxil, cephalexin,cephradine, cephalothin, cephapirin, cefazolin, and the like), secondgeneration cephalosporins (such as cefaclor, cefamandole, cefonicid,cefoxitin, cefotetan, cefuroxime, cefuroxime axetil, cefinetazole,cefprozil, loracarbef, ceforanide, and the like), third generationcephalosporins (such as cefepime, cefoperazone, cefotaxime, ceftizoxime,ceftriaxone, ceftazidime, cefixime, cefpodoxime, ceftibuten, and thelike), other beta-lactams (such as imipenem, meropenem, aztreonam,clavulanic acid, sulbactam, tazobactam, and the like), beta-lactamaseinhibitors (such as clavulanic acid), chloramphenicol, macrolides (suchas erythromycin, azithromycin, clarithromycin, and the like),lincomycin, clindamycin, spectinomycin, polymyxin B, polymixins (such aspolymyxin A, B, C, D, E.sub.1(colistin A), or E.sub.2, colistin B or C,and the like) colistin, vancomycin, bacitracin, isoniazid, rifampin,ethambutol, ethionamide, aminosalicylic acid, cycloserine, capreomycin,sulfones (such as dapsone, sulfoxone sodium, and the like), clofazimine,thalidomide, or any other antibacterial agent that can be lipidencapsulated. Anti-infectives can include antifungal agents, includingpolyene antifungals (such as amphotericin B, nystatin, natamycin, andthe like), flucytosine, imidazoles (such as miconazole, clotrimazole,econazole, ketoconazole, and the like), triazoles (such as itraconazole,fluconazole, and the like), griseofulvin, terconazole, butoconazoleciclopirax, ciclopirox olamine, haloprogin, tolnaftate, naftifine,terbinafine, or any other antifungal that can be lipid encapsulated orcomplexed and pharmaceutically acceptable salts thereof and combinationsthereof. Discussion and the examples are directed primarily towardciprofloxacin but the scope of the application is not intended to belimited to this anti-infective. Combinations of drugs can be used.

A biofilm is any group of microorganisms in which cells stick to eachother on a surface. These adherent cells are frequently embedded withina self-produced matrix of extracellular polymeric substance (EPS).Biofilm extracellular polymeric substance, which is also referred to asslime (although not everything described as slime is a biofilm), is apolymeric conglomeration generally composed of extracellular DNA,proteins, and polysaccharides. Biofilms may form on living or non-livingsurfaces and can be prevalent in natural, industrial and hospitalsettings. The microbial cells growing in a biofilm are physiologicallydistinct from planktonic cells of the same organism, which, by contrast,are single-cells that may float or swim in a liquid medium.

Biofilms have been found to be involved in a wide variety of microbialinfections in the body, by one estimate 80% of all infections.Infectious processes in which biofilms have been implicated includecommon problems such as urinary tract infections, catheter infections,middle-ear infections, formation of dental plaque, gingivitis, coatingcontact lenses, and less common but more lethal processes such asendocarditis, infections in cystic fibrosis, and infections of permanentindwelling devices such as joint prostheses and heart valves. Morerecently it has been noted that bacterial biofilms may impair cutaneouswound healing and reduce topical antibacterial efficiency in healing ortreating infected skin wounds.

As used herein, “Formulation” refers to the liposome-encapsulatedanti-infective, with any excipients or additional active ingredients,either as a dry powder or suspended or dissolved in a liquid.

The terms “subject,” “individual,” “patient,” and “host” are usedinterchangeably herein and refer to any vertebrate, particularly anymammal and most particularly including human subjects, farm animals, andmammalian pets. The subject may be, but is not necessarily under thecare of a health care professional such as a doctor.

A “stable” formulation is one in which the protein or enzyme thereinessentially retains its physical and chemical stability and integrityupon storage and exposure to relatively high temperatures. Variousanalytical techniques for measuring peptide stability are available inthe art and are reviewed in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991), andJones, A. (1993) Adv. Drug Delivery Rev. 10:29-90. Stability can bemeasured at a selected temperature for a selected time period.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

A “disorder” is any condition that would benefit from treatment with theclaimed methods and compositions.

Invention in General

Ciprofloxacin is a well-established and extensively utilizedbroad-spectrum fluoroquinolone antibiotic that is indicated for thetreatment of lower respiratory tract infections, due to, for example, P.aeruginosa, which is common in patients with cystic fibrosis. Theprimary advantage of inhaled antimicrobials is that they targetantibiotic delivery to the area of primary infection and bypassGI-related side effects; however, the poor solubility and bitterness ofthe drug have limited development of a formulation suitable forinhalation. Furthermore, the rapid tissue distribution of ciprofloxacinmeans a short drug residence time in the lung thus limiting therapeuticbenefit over oral or IV drug administration. The liposome-encapsulatedformulations of ciprofloxacin described here decrease the limitationsand improves management of pulmonary infections due to NTM throughimproved biopharmaceutical characteristics and mechanisms such asretention of vesicle size and encapsulation following nebulization,altered drug PK and biodistribution, sustained drug release from thecarrier, enhanced delivery to disease sites including intracellularinfections, whereby the concentration of drug is now higher within theintracellular space.

The invention is not limited to the treatment of patients with NTMinfections. In fact, there are many patients and indications for whichthis therapy may be beneficial, including intracellular infections andparticularly those infections in alveolar macrophages and/or biofilms inthe airways. However, it is particularly useful against mycobacterialinfections because it is effective at killing both replicating andnon-replicating bacteria, which are present in biofilm. As described byMcNabe et al (2012), M. avium forms increasing amounts of biofilm inpresence of antibiotics such as streptomycin and tetracycline, whichstimulate biofilm-related gene expression in the bacterium. Once formed,biofilms are made of two distinct populations of bacteria, sessile, themore resistant phenotype, and planktonic, a susceptible phenotype. Thisit is indeed surprising that inhaled liposomal ciprofloxacin iseffective at killing both populations of bacteria, including sessile,which are more resistant. This should be contrasted to a much weakerefficacy of unencapsulated ciprofloxacin. The difference betweenliposomal and encapsulated ciprofloxacin activity against NTM would belikely to be even greater in vivo because the unencapsulatedciprofloxacin disappears from the airways and the lung much faster thanthe encapsulated ciprofloxacin.

The formulations of the invention may be administered to a patient usinga disposable package and portable, hand-held, battery-powered device,such as the AERx device (U.S. Pat. No. 5,823,178, Aradigm, Hayward,Calif.). Alternatively, the formulations of the instant invention may becarried out using a mechanical (non-electronic) device. Other inhalationdevices may be used to deliver the formulations including conventionaljet nebulizers, ultrasonic nebulizers, soft mist inhalers, dry powderinhalers (DPIs), metered dose inhalers (MDIs), condensation aerosolgenerators, and other systems. The proportion of free ciprofloxacin toencapsulated ciprofloxacin was shown to remain constant afternebulization; i.e., there was no damage to the liposomes duringnebulization that would result in premature release of a portion of theencapsulated antibiotic. This finding is unexpected based upon priorliterature reports (Niven R W and Schreier H, 1990) but ensures that theanimal or human inhaling the aerosol will get a reproducible proportionof free to encapsulated drug depositing throughout the lung.

An aerosol may be created by forcing drug through pores of a membranewhich pores have a size in the range of about 0.25 to 6 microns (U.S.Pat. No. 5,823,178). When the pores have this size the particles whichescape through the pores to create the aerosol will have a diameter inthe range of 0.5 to 12 microns. Drug particles may be released with anair flow intended to keep the particles within this size range. Thecreation of small particles may be facilitated by the use of thevibration device which provides a vibration frequency in the range ofabout 800 to about 4000 kilohertz. Those skilled in the art willrecognize that some adjustments can be made in the parameters such asthe size of the pores from which drug is released, vibration frequency,pressure, and other parameters based on the density and viscosity of theformulation keeping in mind that an object of some embodiments is toprovide aerosolized particles having a diameter in the range of about0.5 to 12 microns.

The liposome formulation may be a low viscosity liquid formulation. Theviscosity of the drug by itself or in combination with a carrier shouldbe sufficiently low so that the formulation can be forced out ofopenings to form an aerosol, e.g., using 20 to 200 psi to form anaerosol preferably having a particle size in the range of about 0.5 to12 microns.

In an embodiment, a low boiling point, highly volatile propellant iscombined with the liposomes of the invention and a pharmaceuticallyacceptable excipient. The liposomes may be provided as a suspension ordry powder in the propellant, or, in another embodiment, the liposomesare dissolved in solution within the propellant. Both of theseformulations may be readily included within a container which has avalve as its only opening. Since the propellant is highly volatile, i.e.has a low boiling point, the contents of the container will be underpressure.

In accordance with another formulation, the ciprofloxacin-containingliposomes are provided as a dry powder by itself, and in accordance withstill another formulation, the ciprofloxacin-containing liposomes areprovided in a solution formulation. The dry powder may be directlyinhaled by allowing inhalation only at the same measured inspiratoryflow rate and inspiratory volume for each delivery. The powder may bedissolved in an aqueous solvent to create a solution which is movedthrough a porous membrane to create an aerosol for inhalation. Anyformulation which makes it possible to produce aerosolized forms ofciprofloxacin-containing liposomes which can be inhaled and delivered toa patient via the intrapulmonary route may be used in connection withthe present invention. Specific information regarding formulations whichcan be used in connection with aerosolized delivery devices aredescribed within Remington's Pharmaceutical Sciences, A. R. Gennaroeditor (latest edition) Mack Publishing Company. Regarding insulinformulations, it is also useful to note the findings of Sciarra et al.,(1976). When low boiling point propellants are used, the propellants areheld within a pressurized canister of the device and maintained in aliquid state. When the valve is actuated, the propellant is released andforces the active ingredient from the canister along with thepropellant. The propellant will “flash” upon exposure to the surroundingatmosphere, i.e., the propellant immediately evaporates. The flashingoccurs so rapidly that it is essentially pure active ingredient which isactually delivered to the lungs of the patient.

Based on the above, it will be understood by those skilled in the artthat a plurality of different treatments and means of administration canbe used to treat a single patient. Thus, patients already receiving suchmedications, for example, as intravenous ciprofloxacin or antibiotics,etc., may benefit from inhalation of the formulations of the presentinvention. Some patients may receive only ciprofloxacin-containingliposome formulations by inhalation. Such patients may be diagnosed ashaving NTM lung infections, or have symptoms of a medical condition,which symptoms may benefit from administration to the patient of anantibiotic such as ciprofloxacin. The formulations of the invention mayalso be used diagnostically.

A patient will typically receive a dose of about 0.01 to 10 mg/kg/day ofciprofloxacin ±20% or ±10%. This dose will typically be administered byat least one, preferably several “puffs” from the aerosol device. Thetotal dose per day is preferably administered at least once per day, butmay be divided into two or more doses per day. Some patients may benefitfrom a period of “loading” the patient with ciprofloxacin with a higherdose or more frequent administration over a period of days or weeks,followed by a reduced or maintenance dose. As NTM is a difficultcondition to treat, patients are expected to receive such therapy over aprolonged period of time.

The invention includes a method of treating non-tuberculous mycobacteriawhereby the formulation of the invention is administered by any knownroute of administration such as injection, inhalation, nasaladministration, orally, and IV infusion. Although a preferred method ofadministration is by inhalation in that the invention is particularlysuited for the treatment of infections in the form of biofilms in thelungs. The formulations of the invention are particular suited for theeradication of infections formed as biofilms in the lung for a number ofreasons. First, the liposomes of the invention are particular resistantto rupture upon aerosolization in that 90% or more, 95% or more, 98% ormore of the liposomes maintain their structural integrity and therebymaintain the drug formulations held within them after being aerosolizedeither by a nebulizer or being moved through the pores of a porousmembrane. After the formulation reaches lung tissue drug dissolved inthe solvent carrier which may be an aqueous carrier at a relatively lowpH such as 6.5 or less, 6.0 or less, 5.5 or less, 5.0 or less drug inthat carrier provides for immediate release and contact with bacteria.Thereafter, the liposomes dissolve or become more permeable and providefor release of formulation encapsulated within the liposomes.Thereafter, the nanocrystals slowly dissolve, when present inside theliposomes. Accordingly, the formulations of the invention can bedelivered on a once a day basis and provided for controlled release ofthe drug such as ciprofloxacin over a long period of time.

Biofilms are resistant to eradication by antibiotics due to a number offactors. First, they are usually surrounded by a dense exopolysaccharidematrix that inhibits the diffusion of some antibiotics, includingaminoglycosides as a class, into the biofilm. Second, the outer layer offaster-growing bacteria cells also “protects” the cells in the interiorof the biofilm from antibiotic exposure. Third, the cells in theinterior of the biofilm are oxygen-deprived and so are slower-growing ordormant and thus intrinsically less sensitive to antibiotic exposure.Finally, there is evidence of the presence of “persister” cells whichare invulnerable to killing and other unknown resistance mechanisms mayalso exist.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor is it intendedto represent that the experiment below is the only experiment performed.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees Centigrade, and pressure is at or nearatmospheric.

Example 1

Manufacture of Encapsulated Ciprofloxacin:

Ciprofloxacin (50 mg/mL) is encapsulated into liposomes consisting ofhydrogenated soy phosphatidyl-choline (HSPC) (70.6 mg/mL), asemi-synthetic fully hydrogenated derivative of natural soy lecithin(SPC), and cholesterol (29.4 mg/mL). The lipid is organized in abilayer, with an average particle size of 75 to 120 nm. The sterilesuspension is suspended in an isotonic buffer (25 mM histidine, 145 mMNaCl at pH 6.0, 300 mOsm/kg) and administered by inhalation. Theseliposomal ciprofloxacin preparations contained approximately 1%unencapsulated ciprofloxacin.

The manufacturing process includes the following steps.

-   1. Preparation of buffers.-   2. Weighing of lipid components.-   3. Dissolution of lipids in solvent (tBuOH:EtOH:dH2O/49:49:2).-   4. Mixing of the solvent solution of lipids with methylamine    sulphate buffer (10% v/v solvent) to form multilamellar vesicles    (MLVs) with encapsulated methylamine sulphate buffer at 30 mg/mL    lipid.-   5. Extrusion through four stacked 80 nm pore size polycarbonate    filters to generate large unilamellar vesicles (LUVs). A second    extrusion pass was performed to generate liposomes with a mean    diameter of ˜100 nm.-   6. Ultrafiltration to concentrate the liposomes to ˜55 mg/mL total    lipid.-   7. Diafiltration against 10 volumes of buffer (145 mM NaCl, 5 mM    histidine, pH 6.0) to remove ethanol and generate a transmembrane pH    gradient.-   8. Determination of the lipid concentration by HPLC.-   9. Heating of the liposome suspension to 50° C. and slow addition of    powdered ciprofloxacin (60% of the total lipid mass) with stirring.    Ciprofloxacin is added incrementally (10% of mass every 4 minutes    over a 40-minute period) and the product incubated at 50° C. for 20    minutes following addition of the last aliquot to allow completion    of the drug loading process.-   10. Diafiltration of the ciprofloxacin loaded liposomes against    3-volumes of 145 mM NaCl, 5 mM acetate, pH 4.0 to remove    unencapsulated ciprofloxacin under conditions in which the free    ciprofloxacin is soluble.-   11. Diafiltration of the ciprofloxacin loaded liposomes against    5-volumes of 145 mM NaCl, 25 mM histidine, pH 6.0 to remove any    remaining un-encapsulated ciprofloxacin, further reducing the    residual solvent levels and exchanging the external buffer for the    desired final product buffer.-   12. Ultrafiltration of the formulation to a ciprofloxacin    concentration of 50 mg/mL (in-process testing required).-   13. Pre-filtration of the liposomes through 0.45/0.2 μm filter    sheets to remove particulates which can clog sterilizing grade    filters. The filters employed are in fact sterilizing grade filters;    however they are employed at elevated pressures not compatible with    their use for sterile filtration.-   14. Redundant filtration through 0.2 μm sterilizing grade filters.-   15. Sample vialing and packaging.

The overall manufacturing scheme is shown in FIG. 1.

Example 2

Activity of Liposomal Ciprofloxacin Against M. avium and M. abscessus inBiofilm.

Preparation of Liposomal Encapsulated Ciprofloxacin and FreeCiprofloxacin:

A solution of unencapsulated, or “free” ciprofloxacin at a concentrationof 20 mg/mL in 10 mM sodium acetate, pH 3.2, was prepared. A preparationof liposomal encapsulated ciprofloxacin was prepared according toExample 1 at 50 mg/mL ciprofloxacin in 25 mM histidine, 145 mM NaCl, pH6.0.

Description of Infection Model:

Formulations containing free ciprofloxacin and liposome encapsulatedciprofloxacin, as well as an empty liposome control and buffer control,were evaluated in a M. avium and M. abscessus biofilm model on a 96 wellplate for 4 days.

Design of Dose-Ranging Study:

One concentration of free or liposomal ciprofloxacin (50 or 100 mcg/mL),empty liposomes as a control, or buffer alone as a negative control wereevaluated in three models. The colonization was reported initially andthen on day 4.

Results:

Table 1 shows the colonization for each arm. Treatment with liposomalciprofloxacin was found to provide a statistically significant effect ineach of these models, while ciprofloxacin alone did not have astatistically significant effect.

TABLE 1 Biofilm Colonization Bacterial Strain Treatment (mcg/mL) Time 0Time: 4 Days P Value M. avium A5 None 3.1 ± 0.7 × 10⁷ 3.4 ± 0.5 × 10⁷ —Ciprofloxacin (100) 2.8 ± 0.7 × 10⁷ p > 0.05 Empty Liposome 3.9 ± 0.5 ×10⁷ p > 0.05 Liposomal Ciprofloxacin (50) 2.0 ± 0.4 × 10⁷ p < 0.05Liposomal Ciprofloxacin (100) 1.1 ± 0.5 × 10⁷ p < 0.05 M. avium 104 None4.3 ± 0.8 × 10⁷ 4.1 ± 0.7 × 10⁷ — Ciprofloxacin (100) 4.0 ± 0.3 × 10⁷p > 0.05 Empty Liposome 4.8 ± 0.6 × 10⁷ p > 0.05 Liposomal Ciprofloxacin(50) 2.6 ± 0.5 × 10⁷ p < 0.05 Liposomal Ciprofloxacin (100) 1.9 ± 0.6 ×10⁷ p < 0.05 M. abscessus 105 None 3.8 ± 0.6 × 10⁷ 3.9 ± 0.8 × 10⁷ —Ciprofloxacin (100) 3.0 ± 0.5 × 10⁷ p > 0.05 Empty Liposome 3.9 ± 0.6 ×10⁷ p > 0.05 Liposomal Ciprofloxacin (50) 3.6 ± 0.4 × 10⁷ p > 0.05Liposomal Ciprofloxacin (100) 1.6 ± 0.5 × 10⁷ p < 0.05

Conclusion:

Only liposomal ciprofloxacin formulations demonstrated killing ofmycobacteria, in the biofilm. This is unprecedented as otherantibiotics, including other liposomal antibiotic formulations, have notdemonstrated the ability to decrease the number of bacteria (i.e., kill)in biofilm models of mycobacteria infections. It is likely that it isdifficult to kill sessile bacteria, e.g., those found in biofilms, dueto a multitude of factors, because of physical protection provided bythe mycobacterial biofilm matrix, as well as phenotypic toleranceagainst environmental stress, including antibiotics (Islam et al.).Thus, treatment of mycobacterial biofilm infections is extremelydifficult (Islam et al.). The results shown in Table 1 are thussurprising because these liposomal ciprofloxacin formulations providedsubstantial and effective killing of dormant or sessile bacteria. Thisis an essential component for treatment of NTM infections which aretypically comprised of replicating and non-replicating (sessile)bacteria. Islam et al. go on to say that multiple mycobacterial species,most notably M. avium, have been found to exist as multicellularcommunities in the environment (i.e., biofilm), as well as in clinicalsettings. The prevalence of these mycobacterial communities in theirnatural habitat can be further appreciated by evidence that theaggregates and pellicles of mycobacteria, routinely observed indetergent-free in vitro cultures, represent a genetically programmeddevelopment of organized, drug-tolerant communities—the key features ofbiofilms.

Example 3

Activity of Liposomal Ciprofloxacin Against M. avium in Macrophages.

Preparation of Liposomal Encapsulated Ciprofloxacin and FreeCiprofloxacin:

A solution of unencapsulated, or “free” ciprofloxacin at a concentrationof 20 mg/mL in 10 mM sodium acetate, pH 3.2, was prepared. A preparationof liposomal encapsulated ciprofloxacin was prepared according toExample 1 at 50 mg/mL ciprofloxacin in 25 mM histidine, 145 mM NaCl, pH6.0. Samples were diluted to the appropriate concentration in the THP-1human macrophage model.

Description of Infection Model:

Formulations containing free ciprofloxacin, liposome encapsulatedciprofloxacin, liposomes containing nanocrystals of ciprofloxacin, aswell as a buffer control, were evaluated in a M. avium bacterial strainin THP-1 human macrophages and infection in the macrophages was measuredafter 4 days.

Design of Study:

One concentration of free or liposomal ciprofloxacin (20 mcg/mL), orbuffer alone as a negative control, were evaluated in two models. Thebacterial colonization was reported initially and then on day 4.

Results:

Table 2 shows the colonization for each arm. Treatment with liposomalciprofloxacin was found to provide a statistically significant effect ineach of these macrophage infection models, while ciprofloxacin alone didnot have a statistically significant effect.

TABLE 2 Colonization of M. avium in macrophages Bacterial StrainTreatment (mcg/mL) Time 0 Time: 4 Days P Value M. avium 101 None  7 ±0.2 × 10⁴ 5.5 ± 0.2 × 10⁵ — Ciprofloxacin (20) 4.9 ± 0.4 × 10⁵ p > 0.05Liposomal Ciprofloxacin (20) 8.1 ± 0.3 × 10³ p < 0.05 LiposomalCiprofloxacin in 9.6 ± 0.4 × 10³ p < 0.05 nanocrystal form M. avium 104None 1.4 ± 0.6 × 10⁴ 6.0 ± 0.5 × 10⁴ — Ciprofloxacin (20) 1.1 ± 0.4 ×10⁴ p > 0.05 Liposomal Ciprofloxacin (20) 3.9 ± 0.5 × 10³ p < 0.05Liposomal Ciprofloxacin in 7.4 ± 0.3 × 10³ p < 0.05 nanocrystal form

Conclusion:

Both liposomal ciprofloxacin formulations have superior activity to freeciprofloxacin against M. avium macrophage infection models.

Example 4

Activity of Liposomal Ciprofloxacin Against M. abscessus in Macrophages.

Preparation of Liposomal Encapsulated Ciprofloxacin and FreeCiprofloxacin:

A solution of unencapsulated, or “free” ciprofloxacin at a concentrationof 20 mg/mL in 10 mM sodium acetate, pH 3.2, was prepared. A preparationof liposomal encapsulated ciprofloxacin was prepared according toExample 1 at 50 mg/mL ciprofloxacin in 25 mM histidine, 145 mM NaCl, pH6.0. Samples were diluted to the appropriate concentration in the THP-1human macrophage model.

Description of Infection Model:

Formulations containing free ciprofloxacin, liposome encapsulatedciprofloxacin, empty liposomes, as well as a buffer control, wereevaluated in a M. abscessus bacterial strain in THP-1 human macrophagesand infection in the macrophages was measured after 4 days.

Design of Dose-Ranging Study:

One concentration of free or liposomal ciprofloxacin (10 or 20 mcg/mL),empty liposomes, or buffer alone as a negative control, were evaluatedin two M. abscessus models. The bacterial colonization was reportedinitially and then on day 4.

Results:

Table 3 shows the colonization for each arm. Treatment with liposomalciprofloxacin was found to provide a statistically significant effect ineach of these macrophage infection models, while ciprofloxacin alone didnot have a statistically significant effect.

TABLE 3 Colonization of M. avium in macrophages Bacterial StrainTreatment (mcg/mL) Time 0 Time: 4 Days P Value M. abscessus 101 None 3.2± 0.4 × 10⁵ 3.6 ± 0.3 × 10⁶ — Ciprofloxacin (10) 3.2 ± 0.4 × 10⁶ p >0.05 Ciprofloxacin (20) 1.9 ± 0.5 × 10⁶ p = 0.07 Empty Liposomes 3.9 ±0.5 × 10⁶ p > 0.05 Liposomal Cip (10) 5.1 ± 0.4 × 10³ p < 0.05 LiposomalCip (20) 3.0 ± 0.3 × 10³ p < 0.05 M. abscessus 102 None 4.1 ± 0.6 × 10⁵3.3 ± 0.5 × 10⁶ — Ciprofloxacin (10) 3.0 ± 0.5 × 10⁶ p > 0.05Ciprofloxacin (20) 1.2 ± 0.3 × 10⁶ p = 0.068 Empty Liposomes 4.6 ± 0.5 ×10⁶ p > 0.05 Liposomal Cip (10) 6.7 ± 0.3 × 10³ p < 0.05 Liposomal Cip(20) 4.2 ± 0.6 × 10³ p < 0.05

Conclusion:

Liposomal ciprofloxacin formulations have superior activity to freeciprofloxacin against M. abscessus macrophage infection models.

Example 5

Activity of Liposomal Ciprofloxacin Against M. abscessus in Macrophages.

Preparation of Liposomal Encapsulated Ciprofloxacin and FreeCiprofloxacin:

A solution of unencapsulated, or “free” ciprofloxacin at a concentrationof 20 mg/mL in 10 mM sodium acetate, pH 3.2, was prepared. A preparationof liposomal encapsulated ciprofloxacin was prepared according toExample 1 at 50 mg/mL ciprofloxacin in 25 mM histidine, 145 mM NaCl, pH6.0. Samples were diluted to the appropriate concentration in the THP-1human macrophage model.

Description of Infection Model:

Formulations containing free ciprofloxacin, liposome encapsulatedciprofloxacin, empty liposomes, as well as a buffer control, wereevaluated in a M. abscessus bacterial strain in THP-1 human macrophagesand infection in the macrophages was measured after 4 days.

Design of Study:

One concentration of free or liposomal ciprofloxacin (200 mcg/mL), emptyliposomes, or buffer alone as a negative control, were evaluated in twoM. abscessus models. The bacterial colonization was reported initiallyand then on day 4.

Results:

Table 4 shows the colonization for each arm. Treatment with liposomalciprofloxacin was found to provide a statistically significant effect ineach of these macrophage infection models, while unencapsulatedciprofloxacin alone did not have a statistically significant effect.

TABLE 4 Colonization of M. avium or M. abscessus in macrophagesBacterial Strain Treatment (mcg/mL) Time 0 Time: 4 Days P Value M. avium104 None 2.9 ± 0.5 × 10⁵ 3.6 ± 0.5 × 10⁶ — Ciprofloxacin (200) 5.0 ± 0.3× 10⁴ p < 0.05 Empty Liposomes 3.9 ± 0.2 × 10⁶ p > 0.05 LiposomalCiprofloxacin (200) 7.1 ± 0.3 × 10² p < 0.05 M. abscessus 101 None 2.6 ±0.4 × 10⁵ 3.2 ± 0.6 × 10⁶ — Ciprofloxacin (200) 4.4 ± 0.6 × 10⁴ p < 0.05Empty Liposomes 4.1 ± 0.2 × 10⁵ p > 0.05 Liposomal Ciprofloxacin (200)7.4 ± 0.3 × 10² p < 0.05

Conclusion:

High concentrations of liposomal ciprofloxacin were more effective thanfree ciprofloxacin in M. abscessus and M. avium macrophage infectionmodels.

Example 6

Activity of Liposomal Ciprofloxacin Against M. avium in C57BL/6 Mice.

Preparation of Liposomal Encapsulated Ciprofloxacin and FreeCiprofloxacin:

A solution of unencapsulated or “free” ciprofloxacin at a concentrationof 20 mg/mL ciprofloxacin HCl (equivalent to 18 mg/mL ciprofloxacin) in10 mM sodium acetate, pH 3.2, was prepared. A preparation of liposomalencapsulated ciprofloxacin was prepared according to Example 1 at 50mg/mL ciprofloxacin HCl (equivalent to 45 mg/mL ciprofloxacin) in 25 mMhistidine, 145 mM NaCl, pH 6.0. A preparation of “Pulmaquin”(Dual-Release Ciprofloxacin for Inhalation, DRCFI) a 1:1volume-to-volume mixture of free ciprofloxacin and liposomalencapsulated ciprofloxacin, with a ciprofloxacin HCl concentration of 35mg/mL). Samples were diluted to the appropriate concentration for dosingthe mice. Pulmaquin thus contains approximately 35 mg/mL ciprofloxacinHCl, with 25 mg/mL in the encapsulated form and 10 mg/mL in theunencapsulated or free form. This is equivalent to 31.5 mg/mLciprofloxacin of which 22.5 mg/mL is encapsulated ciprofloxacin and 9mg/mL is unencapsulated or free ciprofloxacin. The pH of Pulmaquin isbetween 4 and 5, intermediate between the pH of 3.2 for theunencapsulated drug and a pH of 6.0 for the encapsulated drug.

Description of Infection Model:

Formulations containing free ciprofloxacin, liposome encapsulatedciprofloxacin, Pulmaquin, and empty liposome and saline controls wereevaluated in C57BL/6 mice infected via intranasal instillation withMycobacterium avium subsp hominissuis (MAH) MAC104 strain, with a doseof 10⁷, and infection was allowed to develop for 1 week

Design of Study:

One week after infection, treatment via intranasal instillation wasinitiated with either saline, empty liposome control, freeciprofloxacin, liposome encapsulated ciprofloxacin, or Pulmaquin dailyat ciprofloxacin doses of 0.33, 0.66 and 1 mg/kg for 3 weeks. For theempty liposome control, the dose of empty liposome matched the lipidcontent of 1 mg/kg dose of liposome encapsulated ciprofloxacin. Micewere harvested and lungs and spleen plated on 7H10 agar forquantification of the bacterial load in lungs. Ten mice were used perexperimental group.

Results:

Table 5 shows the bacterial loads (colony forming units, CFU) for thelung and spleen. While empty liposomes and free ciprofloxacin had nosignificant effect on the growth of MAH in the lungs of mice comparedwith saline control, administration of 1 mg/kg of liposome encapsulatedciprofloxacin or Pulmaquin was associated with a significant reductionin CFU from (1.06±0.5)×10⁷ to (2.25±0.4)×10⁶ (−79%) and (2.47±0.6)×10⁶(−77%), respectively (p<0.05 for both vs saline or empty liposomes).Treatment with 0.33 mg/kg and 0.67 mg/kg liposome encapsulatedciprofloxacin or Pulmaquin also resulted in significant reduction of CFUin the lungs (−37 to −67%).

TABLE 5 Bacterial Loads (CFU) in Lungs and Spleen in MAH Infected MiceTime Treatment Dose (mg/kg) Lung CFU Spleen CFU Baseline No Treatment 2.0 ± 0.4 × 10⁶ 5.39 ± 0.4 × 10⁴ 3 weeks Saline 1.06 ± 0.5 × 10⁷ 4.72 ±0.3 × 10⁵ 3 weeks Empty liposomes 1 2.51 ± 0.4 × 10⁷ 6.27 ± 0.4 × 10⁵ 3weeks Free Ciprofloxacin 1 8.65 ± 0.4 × 10⁶* 6.47 ± 0.4 × 10⁵ 0.67 1.04± 0.4 × 10⁷ 5.99 ± 0.6 × 10⁵ 0.33 2.64 ± 0.4 × 10⁷ 7.38 ± 0.3 × 10⁵ 3weeks Liposomal Ciprofloxacin 1 2.25 ± 0.4 × 10⁶** 3.13 ± 0.4 × 10⁵ 0.673.72 ± 0.5 × 10⁶* 7.25 ± 0.3 × 10⁵ 0.33 5.84 ± 0.3 × 10⁶* 8.28 ± 0.4 ×10⁵ 3 weeks Pulmaquin 1 2.47 ± 0.6 × 10⁶** 6.14 ± 0.4 × 10⁵ 0.67 3.49 ±0.4 × 10⁶* 7.02 ± 0.3 × 10⁵ 0.33 6.71 ± 0.3 × 10⁶* 8.30 ± 0.5 × 10⁵ *p <0.05 than saline/empty **p < 0.05 than free ciprofloxacin

Conclusion:

Three-week intranasal treatment of mice with lung infections from MAHwith liposome encapsulated ciprofloxacin or Pulmaquin resulted insignificant reduction of MAH load in the lungs.

Example 7

Activity of Liposomal Ciprofloxacin Against M. abscessus in Mice.

Preparation of Liposomal Encapsulated Ciprofloxacin and FreeCiprofloxacin:

A solution of unencapsulated or “free” ciprofloxacin HCl at aconcentration of 20 mg/mL (equivalent to 18 mg/mL ciprofloxacin) in 10mM sodium acetate, pH 3.2, was prepared. A preparation of liposomalencapsulated ciprofloxacin was prepared according to Example 1 at 50mg/mL ciprofloxacin HCl (equivalent to 45 mg/mL ciprofloxacin) in 25 mMhistidine, 145 mM NaCl, pH 6.0. A preparation of “Pulmaquin”(Dual-Release Ciprofloxacin for Inhalation, DRCFI) a 1:1volume-to-volume mixture of free ciprofloxacin and liposomalencapsulated ciprofloxacin, with a ciprofloxacin HCl concentration of 35mg/mL, equivalent to 31.5 mg/mL ciprofloxacin). Samples were diluted tothe appropriate concentration for dosing the mice.

Description of Infection Model:

Formulations containing free ciprofloxacin, liposome encapsulatedciprofloxacin, Pulmaquin, and empty liposome and saline controls wereevaluated in C57 BL/6J-lysbg-J/J mice infected via intranasalinstillation with Mycobacterium abscessus strain MA26 with a dose of(5.4±0.3)×10⁷, which is a clinical isolate from a patient at theNational Institutes of Health, in 50 μL of buffer (Hank Balanced SaltSolution) and infection was allowed to develop for 1 week.

Design of Study:

One week after infection, treatment via intranasal instillation wasinitiated with either saline, empty liposome control, freeciprofloxacin, liposome encapsulated ciprofloxacin, or Pulmaquin dailywith a ciprofloxacin dose of 1 mg/kg for 3 or 6 weeks. For the emptyliposome control, the dose of empty liposome matched the lipid contentof 1 mg/kg dose of liposome encapsulated ciprofloxacin. Mice wereharvested and lungs and spleen plated on 7H10 agar for quantification ofthe bacterial load in lungs. Ten mice were used per experimental group.

Results:

Table 6 shows the bacterial loads (colony forming units, CFU) for thelung and spleen. While empty liposomes and free ciprofloxacin had nosignificant effect on the growth of M abscessus in the lungs of micecompared with the untreated control, administration of liposomeencapsulated ciprofloxacin or Pulmaquin for 6 weeks was associated withsignificant reductions in CFU from untreated at 6 weeks (5.4±0.6)×10⁵ to(1.4±0.5)×10³ (−99.7%) and (3.0±0.4)×10³ (−99.4%), respectively (p<0.05for both vs. untreated control). Treatment for 3 weeks with liposomeencapsulated ciprofloxacin or Pulmaquin was also associated withsignificant reductions in CFU from untreated at 6 weeks (5.4±0.6)×10⁵ to(2.6±0.6)×10⁴ (−95.2%) and (2.1±0.4)×10⁴ (−96.1%), respectively (p<0.05for both vs. untreated control).

TABLE 6 Bacterial Loads (CFU) in Lungs and Spleen in M abscessusInfected Mice Treatment Weeks Lung CFU Spleen CFU Baseline 1 2.6 ± 0.4 ×10⁶ 1.5 ± 0.5 × 10⁴ Untreated 6 5.4 ± 0.6 × 10⁵ 7.3 ± 0.4 × 10³ EmptyLiposomes 3 4.8 ± 0.5 × 10⁵ 5.7 ± 0.3 × 10⁴ Empty Liposomes 6 3.6 ± 0.3× 10⁵ 3.5 ± 0.4 × 10⁴ Free Ciprofloxacin 3 5.3 ± 0.3 × 10⁵ 5.5 ± 0.4 ×10⁴ Free Ciprofloxacin 6 4.0 ± 0.4 × 10⁵ 2.7 ± 0.3 × 10⁴ LiposomalCiprofloxacin 3 2.6 ± 0.6 × 10⁴* 2.5 ± 0.3 × 10⁴ Liposomal Ciprofloxacin6 1.4 ± 0.5 × 10³* 9.1 ± 0.3 × 10³ Pulmaquin 3 2.1 ± 0.4 × 10⁴* 2.8 ±0.4 × 10⁴ Pulmaquin 6 3.0 ± 0.4 × 10³* 8.2 ± 0.5 × 10³ *p < 0.05compared to controls

Conclusion:

Three- or six-week intranasal treatment of mice with lung infectionsfrom M abscessus with liposome encapsulated ciprofloxacin or Pulmaquinresulted in significant reduction of bacterial load in the lungs.

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What is claimed is:
 1. An aerosolized dose of a formulation comprised ofaerosolized particles having an aerodynamic diameter in a range of from1 micron to 12 microns; wherein the aerosolized formulation comprises: aliquid carrier comprising ciprofloxacin at a concentration of 20 mg/mLto 80 mg/mL of ciprofloxacin in solution, liposome unencapsulatedciprofloxacin in solution; and ciprofloxacin as nanocrystalsencapsulated inside the liposomes, wherein nanocrystals in the liposomeshave a dimension of 10 nanometers.
 2. The aerosolized formulation ofclaim 1, wherein, the aerosolized particles have an aerodynamic diameterof two microns to eight microns, the liposomes have a diameter of lessthan 1 micron, and nanocrystals in the liposome have a dimension of 100nanometers; and wherein the ciprofloxacin is present in the solution ata concentration of 40 mg/mL to 60 mg/mL; and further wherein theliposomes are unilamellar and maintain structural integrity at a levelof 90% or more after aerosolizing.
 3. The aerosolized formulation ofclaim 1, wherein the liposomes are comprised of a cryopreservative and asurfactant, and wherein the liposomes are characterized such that 95% ormore of the liposomes maintain structural integrity and continue toencapsulate nanocrystals of ciprofloxacin in the aerosolizedformulation.
 4. The aeorosolized formulation of claim 3, wherein theliposomes are characterized such that 98% or more of the liposomesmaintain structural integrity and continue to encapsulate nanocrystalsof ciprofloxacin in the aerosolized formulation.
 5. A pharmaceuticalformulation, comprising: a pharmaceutically acceptable carrier; andliposomes in the carrier wherein the liposomes comprise: a lipidbilayer; a cryopreservative; and nanocrystals precipitated out of asolution of a pharmaceutically active anti-infective drug, and thenanocrystals are surrounded by the lipid bilayer wherein thenanocrystals have dimensions of 100 nm or less.
 6. The formulation ofclaim 5, wherein the cryopreservation is selected from the groupconsisting of sucrose and trehalose.
 7. The formulation of claim 5,further comprising: a pharmaceutically active anti-infective drugdissolved in the carrier.
 8. The formulation of claim 5, wherein thecarrier is a liquid anti-infective drug in which the liposomes aredispersed.
 9. The formulation of claim 7, wherein the anti-infectivedrug is selected from the group comprising a quinolone, a sulfonamide,an aminoglycoside, a tetracycline, para-aminobenzoic acid, adiaminopyrimidine, a beta-lactam, a beta-lactam and a beta-lactamaseinhibitor, chloramphenicol, a macrolide, lincomycin, clindamycin,spectinomycin, polymyxin B, colistin, vancomycin, bacitracin, isoniazid,rifampin, ethambutol, ethionamide, aminosalicylic acid, cycloserine,capreomycin, a sulfone, clofazimine, thalidomide, polyene antifungal,flucytosine, imidazole, triazole, griseofulvin, terconazole,butoconazole ciclopirax, ciclopirox olamine, haloprogin, tolnaftate,naftifine, terbinafine and combinations thereof.
 10. The formulation ofclaim 5, wherein the lipid bilayer is comprised of a lipid selected fromthe group consisting of fatty acids; lysolipids; sphingolipids;sphingomyelin; glycolipids; glucolipids; glycosphingolipids; palmiticacid; stearic acid; arachidonic acid; oleic acid; lipids bearingsulfonated mono-, di-, oligo- or polysaccharides; lipids with ether andester-linked fatty acids, polymerized lipids, diacetyl phosphate,stearylamine, cardiolipin, phospholipids, synthetic phospholipids withasymmetric acyl chains; and lipids bearing a covalently bound polymer.11. The formulation of claim 5, wherein the liposome comprises aphospholipid selected from the group consisting of phosphatidylcholines,lysophosphatidylcholines, phosphatidylethanolamines,phosphatidylinositols, phosphatidylglycerols, phosphatidic acid,phosphatidylserines, and mixtures thereof; wherein said phospholipid isprovided in admixtures with a modifying agent selected from the groupconsisting of cholesterols, stearyl amines, stearic acid, tocopherols,and mixtures thereof; and wherein the liposomes are unilamellar ormultilamellar.
 12. The aerosolized formulation of claim 1, wherein: theliposomes comprise a cryopreservative and a surfactant and have anaverage particle size of about 75 nm to about 120 nm and areunilamellar.
 13. The aerosolized formulation of claim 1, wherein: theliposomes are comprised of cholesterol and hydrogenated soyphosphatidyl-choline (HSPC) at a ratio of about 30 to 70 (plus or minus10%); and wherein the formulation further comprising an excipientsuitable for pulmonary delivery comprised of sodium acetate and anisotonic buffer; and further wherein the liposomes are characterizedsuch that 90% or more of the liposomes maintain integrity whenaerosolized and after contacting lung tissue provide a ciprofloxacinrelease rate of 0.5% to 10% per hour.
 14. The aerosolized formulation ofclaim 1, wherein: the liposomes are characterized such that 95% or moreof the liposomes maintain integrity when aerosolized and aftercontacting lung tissue provide a ciprofloxacin release rate of 1% to 8%per hour.
 15. The aerosolized formulation of claim 1, wherein: theliposomes are further comprised of 0.1 to 0.3% polysorbate 20, and 200to 400 mg/mL sucrose.
 16. The aerosolized formulation of claim 1 whereinthe liposomes comprise cholesterol and hydrogenated soyphosphatidyl-choline (HSPC) at a ratio of 29.4 to 70.6, and areunilamellar and wherein the liposomes are characterized such that 98% ormore of the liposomes maintain integrity when aerosolized, and provide aciprofloxacin release rate of 2% to 6% per hour.
 17. An aerosolized doseof a formulation comprised of aerosolized particles having anaerodynamic diameter in a range of from 1 micron to 12 microns; whereinthe aerosolized formulation comprises: a liquid carrier comprisingciprofloxacin at a concentration of 20 mg/mL to 80 mg/mL ofciprofloxacin in solution, liposome unencapsulated ciprofloxacin insolution; and ciprofloxacin as nanocrystals encapsulated inside theliposomes; wherein the liposomes are comprised of a cryopreservative anda surfactant, and wherein the liposomes are characterized such that 95%or more of the liposomes maintain structural integrity and continue toencapsulate nanocrystals of ciprofloxacin in the aerosolizedformulation.
 18. The aerosolized formulation of claim 17, wherein, theaerosolized particles have an aerodynamic diameter of two microns toeight microns, the liposomes have a diameter of 20 nanometers to 1micron, nanocrystals in the liposomes have a dimension of 10 nanometers;and wherein the ciprofloxacin is present in the solution at aconcentration of 40 mg/mL to 60 mg/mL; and further wherein the liposomesare unilamellar and maintain structural integrity at a level of 98% ormore after aerosolizing.
 19. An aerosolized dose of a formulationcomprised of aerosolized particles having an aerodynamic diameter in arange of from 1 micron to 12 microns; wherein the aerosolizedformulation comprises: a liquid carrier comprising ciprofloxacin at aconcentration of 20 mg/mL to 80 mg/mL of ciprofloxacin in solution,liposome unencapsulated ciprofloxacin in solution; and ciprofloxacin asnanocrystals precipitated out of a solution of ciprofloxacin whichnanocrystals are encapsulated inside the liposomes.
 20. The aerosolizedformulation of claim 19, wherein, the aerosolized particles have anaerodynamic diameter of two microns to eight microns, the liposomes havea diameter of 20 nanometers to 1 micron, and the nanocrystals havedimensions of 10 nanometers or more; and wherein the ciprofloxacin ispresent in the solution at a concentration of 40 mg/mL to 60 mg/mL; andfurther wherein the liposomes are unilamellar and maintain structuralintegrity at a level of 90% or more after aerosolizing.
 21. Theaerosolized formulation of claim 19, wherein the liposomes are comprisedof a cryopreservative and a surfactant, and wherein the liposomes arecharacterized such that 95% or more of the liposomes maintain structuralintegrity and continue to encapsulate nanocrystals of ciprofloxacin inthe aerosolized formulation.
 22. An aerosolized dose of a formulationcomprised of aerosolized particles having an aerodynamic diameter in arange of from 1 micron to 12 microns; wherein the aerosolizedformulation comprises: a liquid carrier comprising ciprofloxacin at aconcentration of 20 mg/mL to 80 mg/mL of ciprofloxacin in solution,liposome unencapsulated ciprofloxacin in solution; and ciprofloxacin asnanocrystals encapsulated inside the liposomes; wherein the liposomesare comprised of cholesterol and hydrogenated soy phosphatidyl-choline(HSPC) of about 30 to 70 (plus or minus 10%); and wherein theformulation further comprising an excipient suitable for pulmonarydelivery comprised of sodium acetate and an isotonic buffer; and furtherwherein the liposomes are characterized such that 90% or more of theliposomes maintain integrity when aerosolized and after contacting lungtissue provide a ciprofloxacin release rate of 0.5% to 10% per hour. 23.The aerosolized formulation of claim 22, wherein, the aerosolizedparticles have an aerodynamic diameter of two microns to eight microns,the liposomes have a diameter of 20 nanometers to 1 micron, and thenanocrystals have dimensions of 10 nanometers or more; and wherein theciprofloxacin is present in the solution at a concentration of 40 mg/mLto 60 mg/mL; and further wherein the liposomes are unilamellar andmaintain structural integrity at a level of 90% or more afteraerosolizing.
 24. The aerosolized formulation of claim 22, wherein theliposomes are comprised of a cryopreservative and a surfactant, andwherein the liposomes are characterized such that 95% or more of theliposomes maintain structural integrity and continue to encapsulatenanocrystals of ciprofloxacin in the aerosolized formulation.
 25. Theaeorosolized formulation of claim 23, wherein the liposomes arecharacterized such that 98% or more of the liposomes maintain structuralintegrity and continue to encapsulate nanocrystals of ciprofloxacin inthe aerosolized formulation.
 26. The aerosolized formulation of claim22, wherein: the liposomes are characterized such that 95% or more ofthe liposomes maintain integrity when aerosolized and after contactinglung tissue provide a ciprofloxacin release rate of 1% to 8% per hour.27. The aerosolized formulation of claim 22, wherein the liposomescomprise cholesterol and hydrogenated soy phosphatidyl-choline (HSPC) ata ratio of 29.4 to 70.6, and are unilamellar and wherein the liposomesare characterized such that 98% or more of the liposomes maintainintegrity when aerosolized, and provide a ciprofloxacin release rate of2% to 6% per hour.
 28. The aerosolized formulation of claim 22, wherein:the liposomes are further comprised of 0.1 to 0.3% polysorbate 20, and200 to 400 mg/mL sucrose.